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PubMed2005

Dynamics of membranes driven by actin polymerization.

A motile cell, when stimulated, shows a dramatic increase in the activity of its membrane, manifested by the appearance of dynamic membrane structures such as lamellipodia, filopodia, and membrane ruffles. The external stimulus turns on membrane bound activators, like Cdc42 and PIP2, which cause increased branching and polymerization of the actin cytoskeleton in their vicinity leading to a local protrusive force on the membrane. The emergence of the complex membrane structures is a result of the coupling between the dynamics of the membrane, the activators, and the protrusive forces. We present a simple model that treats the dynamics of a membrane under the action of actin polymerization forces that depend on the local density of freely diffusing activators on the membrane. We show that, depending on the spontaneous membrane curvature associated with the activators, the resulting membrane motion can be wavelike, corresponding to membrane ruffling and actin waves, or unstable, indicating the tendency of filopodia to form. Our model also quantitatively explains a variety of related experimental observations and makes several testable predictions.

Actins↗

PubMed2005

Regulation of cortactin/dynamin interaction by actin polymerization during the fission of clathrin-coated pits.

Separation of clathrin-coated pits from the plasma membrane, a key event during endocytosis, is thought to be driven by dynamin and the actin cytoskeleton. However, the mechanism for the actin-mediated endocytosis remains elusive. RNA interference-mediated suppression of cortactin, an F-actin binding protein that promotes Arp2/3 complex-mediated actin polymerization, effectively blocked transferrin uptake. Depletion of cortactin in brain cytosol inhibited formation of clathrin-coated vesicles by 70% as analyzed in a cell-free system. Interestingly, the interaction between cortactin and dynamin 2 in cells was dependent on actin polymerization and was attenuated upon cell exposure to cytochalasin D as analyzed by immunofluorescence and immunoprecipitation. Moreover, a cortactin mutant deficient in Arp2/3 binding colocalized less efficiently with dynamin 2 and inhibited the uptake of transferrin. The effect of actin polymerization on the interaction between cortactin and the dynamin proline-rich domain (PRD) was further evaluated under a condition for actin polymerization in vitro. Cortactin binds to the dynamin PRD with an equilibrium dissociation constant of 81 nM in the presence of the Arp2/3 complex and actin, and 617 nM in the absence of actin polymerization. Taken together, these data demonstrate that Arp2/3-mediated actin polymerization regulates the accessibility of cortactin to dynamin 2 and imply a novel mechanism by which cortactin and dynamin drive the fission of clathrin-coated pits in an actin polymerization dependent manner.

Actin Cytoskeleton↗

PubMed1999

Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle.

1. The role of actin polymerization in the regulation of smooth muscle contractility was investigated in canine trachealis muscle strips. The effect of contractile activation on the content of monomeric globular (G)-actin was estimated by the method of DNase I inhibition. The G-actin content was 30 % lower in extracts of muscle strips activated with 10-4 M acetylcholine (ACh) than in extracts from unstimulated muscle strips. The decrease in G-actin in response to contractile stimulation was prevented by latrunculin-A, an agent that prevents actin polymerization by binding to G-actin monomers. 2. The inhibition of actin polymerization by latrunculin-A markedly depressed force development in response to ACh but had no effect on ACh-induced myosin light chain (MLC) phosphorylation. Latrunculin also suppressed the length sensitivity of force during ACh-induced isometric contractions. The actin-capping agent cytochalasin-D also markedly inhibited force and caused only a slight decrease in MLC phosphorylation. Cytochalasin-D also inhibited force in alpha-toxin-permeabilized muscle strips that were activated either by Ca2+ or by ACh at constant pCa. No disorganization of smooth muscle cell ultrastructure was detected by electron microscopy or by immunofluorescence microscopy of muscles treated with either agent. 3. The results suggest that the polymerization of actin is stimulated by the contractile activation of tracheal smooth muscle and that this actin polymerization contributes directly to force development. In addition, actin filament remodelling contributes to the length sensitivity of tracheal smooth muscle contractility.

Acetylcholine↗

PubMed2005

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins.

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.

Actins↗

PubMed1987

Abnormality in actin polymerization associated with defective chemotaxis in neutrophils from neonates.

In an attempt to determine the mechanism of the profound defect in chemotaxis observed in the neutrophils of human neonates, we have examined the generation of polymerized or filamentous actin (F actin) following stimulation of the cells with chemotactic factors. We have also examined the changes in the intracellular levels of free calcium in neonatal neutrophils and compared the results with those in adult neutrophils. Following exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or zymosan-activated serum (ZyAS), neutrophils from adult donors showed an increase in intracellular free calcium, as determined by Quin 2/AM fluorescence, and in actin polymerization (45-55%), as measured by nitrobenzoxadiazole phallicidin fluorescence. These responses were abolished by preincubation with the calcium antagonist verapamil (0.1 mM), which inhibits both calcium influx and release from intracellular stores. In marked contrast to the results obtained with neutrophils from adults, neutrophils from newborn infants, which have defective chemotactic responses, failed to generate F actin following FMLP or ZyAS stimulation and developed significantly lower levels of free intracellular calcium.

Actins↗

PubMed1984

Decondensation of human spermatozoal chromatin by nuclear actin polymerization.

A critical relationship exists between nuclear actin polymerization and decondensation of sperm chromatin. Characteristic decondensation phenomena were brought about by the protein S-1 of heavy meromyosin of rabbit skeletal muscle in sperm that had undergone the acrosome reaction. Sperm treated with only calcium or only ionophore were not affected by the S-1 trigger, and the nucleus remained in the condensed state. Since S-1 specifically binds to actin, it was possible to demonstrate this phenomenon at the ultrastructural level. Maybe there are switches that operate at different steps for events leading to syngamy and fertilization. No switch can be operative until the preceding event has prepared the sperm for entering the next phase. The operations are performed in a perfect sequential order. This investigation leads to the conclusion that decondensation of sperm chromatin is brought about by nuclear actin polymerization.

Acrosome↗

PubMed2006

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate.

Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration of "metavanadate" solution that contains ortho and metavanadate species, as observed by combining kinetic with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the "decavanadate" interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.

Actins↗

PubMed1988

Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle.

We report here on the purification and characterization of a new 25-kDa inhibitor of actin polymerization from turkey gizzard smooth muscle. The protein was purified by chromatography on DEAE-cellulose and hydroxyapatite, as well as by affinity chromatography on an immobilized-antibody column. The purified polypeptide reduced the low-shear viscosity of actin, apparently due to its inhibitory effect on actin polymerization. We demonstrate that this protein is largely responsible for the apparent inhibitory activity previously reported to be associated with smooth muscle vinculin preparations. Three independent monoclonal antibodies prepared against the 25-kDa inhibitor of actin polymerization can effectively adsorb the inhibiting activity of actin polymerization from the crude vinculin preparation or inhibit it. We also show here that the 25-kDa inhibitor of actin polymerization tends to undergo dimerization when maintained in non-reducing buffers, concomitant with the loss of its inhibitory activity. Immunohistochemical labeling of frozen sections, as well as immunoblotting analyzes, indicated that the 25-kDa inhibitor of actin polymerization is particularly enriched in smooth muscle cells and that its distribution is apparently homogenous throughout the cytoplasm showing no apparent enrichment in the vinculin-rich dense plaques located along the endofacial surface of the plasma membrane.

Actins↗

PubMed1985

Effects of temperature on actin polymerized by Ca2+. Direct evidence of fragmentation.

When the temperature is lowered from 20 to 4 degrees C, the specific viscosity of actin polymerized in the presence of either 4 mM-CaCl2 or 2 mM-MgCl2, but not of actin polymerized in the presence of 90 mM-KCl, is decreased by 50% in the absence of free ATP. Addition of ATP restores the viscosity of the actin polymerized by Mg2+, but not that of actin polymerized by Ca2+, to the original value. The effect of temperature on actin polymerized in the presence of Ca2+ is due to (a) polymer-into-monomer conversion, (b) latero-lateral aggregation of filaments, and (c) fragmentation of the filaments. Fragmentation, as demonstrated by fractional centrifugation and electron microscopy, was the most important of these.

Actins↗

PubMed2004

Interleukin-5, interleukin-3 and granulocyte-macrophage colony-stimulating factor prime actin-polymerization in human eosinophils: a study with hypodense and normodense eosinophils from patients with atopic dermatitis.

Characteristic features of atopic diseases (AD) are immigration and local activation of eosinophils. Reorganization of the cytoskeleton modulates the function of leukocytes and is a prerequisite for the motility response. In this work, the regulation of actin polymerization has been investigated by flow cytometry using NBD-phallacidin and right angle light scatter measurements in purified eosinophils isolated from patients with atopic dermatitis and normal individuals. Stimulation of eosinophils with chemotaxins such as complement fragment C5a (C5a), CC chemokine RANTES/ CCL5 and platelet activating factor (PAF) induced a reversible polymerization of actin. Normodense eosinophils purified from patients with AD showed a decreased chemotaxin-induced actin response as compared to normodense eosinophils from healthy subjects and hypodense eosinophils from patients. Stimulation of eosinophils with Th2-cytokines such as interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF) did not exert a significant effect on actin polymerization. However, pretreatment with IL-3, IL-5 or GM-CSF potentiated the chemotaxin-induced actin polymerization and graded the differential responsiveness between normodense and hypodense eosinophils. We demonstrate a different actin responsiveness in eosinophils from atopic patients and healthy subjects which could be overcome by modulating effects of Th2-cytokines.

Actins↗

PubMed1997

Actin polymerization in neutrophils from patients affected by myelodysplastic syndromes--a flow cytometric study.

In this study F-actin polymerization in neutrophils from 21 patients affected by myelodysplastic syndromes (MDS) was evaluated by means of a flow cytometric assay. Neutrophils were stimulated with formyl-methionyl-leucyl-phenylanaline (fMLP; 10(-8) M final concentration) for 15, 30, 60 and 120 sec, and F-actin content was determined using fluorescein-isothiocyanate phallacidin as a specific probe. Eight normal subjects were studied as controls. We found that F-actin polymerization was defective in ten patients, with very impaired values after 60 and 120 sec of stimulation with fMLP. The remaining 11 patients showed a prevalent neutrophil population with normal F-actin polymerization and neutrophil sub-populations with either defective or undetectable F-actin polymerization. In the first group, patients with very poor prognosis (refractory anemia with excess blasts, refractory anemia with excess blasts in leukemic transformation, trisomy 8, multiple karyotypic abnormalities) were present, although patients with aberrations of karyotype were present in the second group. It is possible that defects in neutrophil F-actin polymerization may be responsible for neutrophil dysfunction, which has frequently been observed in MDS.

Actins↗

PubMed1997

Thyroid hormone-regulated actin polymerization in brain.

Thyroid hormones play an important role in the growth and development of the brain. Central to the proper integration of neuronal circuitry is the ability of the growing neurite to interpret guidance cues during its migration. The action cytoskeleton is especially rich in the growth cone, and is a likely target for thyroid hormone regulation. This brief review summarizes work showing that thyroxine, but not T3, dynamically regulates the polymerization of the actin cytoskeleton in astrocytes. The ability of T4 to enhance actin polymerization, without directly affecting gene expression, has a profound effect on the ability of the cell to interact with laminin, the major extracellular matrix protein in the developing brain. T4 also regulates the formation of key cell contacts with extracellular matrix guidance cues. These processes are likely to participate in thyroid hormone's regulation of brain development.

Actins↗

PubMed1998

A mathematical model for ligand/receptor/G-protein dynamics and actin polymerization in human neutrophils.

A mathematical model is proposed for describing the dynamics of the chemotactic peptide-stimulated actin polymerization response in human neutrophils. The response pathway utilizes the guanine nucleotide binding protein (G-protein) signal transduction cascade common to many receptor systems and allows adaptation in the continued presence of ligand. The development of such a model is an important first step toward understanding, predicting, and ultimately manipulating neutrophil responses. The model is divided into two parts, ligand/receptor/G-protein dynamics and the actin polymerization mechanism. Fast (receptor precoupled to G-protein) and slow (free receptor) signaling pathways involving ligand/receptor/G-protein interactions produce an activated signaling molecule. The actin polymerization mechanisms utilizes an actin binding protein which complexes with actin monomer and inhibits polymerization in an unstimulated cell. During stimulation, the activated signaling molecule enhances the dissociation of monomer/binding protein complexes, allowing the actin polymerization response to occur. The fast and slow signaling pathways are predicted to have different roles in controlling the time course of this actin polymerization. Additionally, precoupled receptors are predicted to have a larger ligand association rate constant than non-precoupled (free) receptors. Model simulations agree with many of the experimentally observed characteristics of both the stimulated F-actin response and ligand/receptor binding kinetics for both the fluorescent peptide ligand CHO-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine-fluorescein (CHO-NLFNTK-fl) and the non-fluorescent peptide ligand CHO-methionyl-leucyl-phenylalanine (CHO-MLF).

Actins↗

PubMed1995

The actin-polymerization protein from Listeria ivanovii is a large repeat protein which shows only limited amino acid sequence homology to ActA from Listeria monocytogenes.

Within infected eukaryotic cells the two pathogenic Listeria species, L. monocytogenes and L. ivanovii, induce polymerization of cellular actin and the formation of a propulsive actin tail at one bacterial pole. For L. monocytogenes it has been shown that the product of the listerial actA gene is required for this process which is regarded as a model for actin-based motility. We have now cloned and sequenced a functionally analogous gene from L. ivanovii; its product, as deduced from the DNA sequence, is considerably larger (108 kDa) than L. monocytogenes ActA (67 kDa) and shares only a limited amino acid sequence homology (46% similarity on average) with the latter protein. This is the first example of a virulence gene product from L. ivanovii which is significantly different from its L. monocytogenes counterpart. Comparison of the two ActA proteins gives new insight into the structure of this class of actin-polymerization proteins, in particular with respect to their proline-rich repeat region.

Amino Acid Sequence↗

PubMed1996

Effects of synaptic vesicles on actin polymerization.

We have analyzed the effects of synaptic vesicles on actin polymerization by using a time-resolved spectrofluorometric assay. We have found that synaptic vesicles have complex effects on the kinetics of actin polymerization, which vary depending on whether the synaptic vesicle-specific phosphoprotein synapsin I is absent or present on their membrane. Synapsin I bound either to synaptic vesicles or to pure phospholipid vesicles exhibits phosphorylation-dependent actin-nucleating activity. Synaptic vesicles depleted of endogenous synapsin I decrease the rate and the final extent of actin polymerization, an effect which is not observed with pure phospholipid vesicles. Thus, the state of association of synapsin I with synaptic vesicles, which is modulated by its state of phosphorylation, may affect actin assembly and the physico-chemical characteristics of the synaptic vesicle microenvironment.

Actins↗

PubMed1997

Effect of major surgery on neutrophil chemotaxis and actin polymerization in neonates and children.

The authors have examined the effect of major surgery in neonates and older children on neutrophil (PMN) chemotaxis and on actin polymerization, an essential early step in PMN movement. Isolated PMNs from the following subjects were studied: healthy adult volunteers (n = 28), healthy newborns (n = 21), newborns undergoing major surgery (n = 7), and older infants and children undergoing major surgery (n = 14). Chemotaxis was measured by a micropore filter assay, and actin polymerization was measured by flow cytometry. Blood samples from surgical patients were obtained preoperatively, hourly during the procedure, immediately postoperatively, and 48 hours after surgery. Mean preoperative newborn PMN chemotaxis was similar to that of healthy newborn PMN, and mean preoperative PMN chemotaxis in children was similar to that of healthy adults. There were no significant alterations in PMN chemotaxis during or after major surgery in neonates or children. Peak PMN actin polymerization, after stimulation with formyl methionyl leucyl phenylalanine (FMLP) (10 nm), was significantly diminished in healthy neonates compared with adults (P < .005). Preoperative surgical neonates had similar peak PMN actin polymerization levels to those of healthy newborns, and older preoperative children had similar levels to adults. PMN actin polymerization did not significantly change during or after major surgery. Despite reductions in PMN chemotaxis and actin polymerization in healthy neonates, there is no further impairment of these PMN functions during or after major surgery. Our data suggest that PMN chemotactic function is resistant to the stress of uncomplicated major surgery in neonates and children.

Actins↗

PubMed2005

Actin polymerization promotes the reversal of streaming in the apex of pollen tubes.

Actin polymerization is important in the control of pollen tube growth. Thus, treatment of pollen tubes with low concentrations of latrunculin B (Lat-B), which inhibits actin polymerization, permits streaming but reversibly blocks oscillatory growth. In the current study, we employ Jasplakinolide (Jas), a sponge cyclodepsipeptide that stabilizes actin microfilaments and promotes polymerization. Uniquely, Jas (2 microM) blocks streaming in the shank of the tube, but induces the formation of a toroidal-shaped domain in the swollen apex, of which longitudinal optical sections exhibit circles of motion. The polarity of this rotary motion is identical to that of reverse fountain motility in control pollen tubes, with the forward direction occurring at the edge of the cell and the rearward direction in the cell interior. Support for the idea that actin polymerization in the apical domain contributes to the formation of this rotary motility activity derives from the appearance therein of aggregates and flared cables of F-actin, using immunofluorescence, and by the reduction in G-actin as indicated with fluorescent DNAse. In addition, Jas reduces the tip-focused Ca2+ gradient. However, the alkaline band appears in the swollen apex and is spatially localized with the reverse fountain streaming activity. Taken together, our results support the idea that actin polymerization promotes reversal of streaming in the apex of the lily pollen tube.

Actin Cytoskeleton↗

PubMed1982

Actin polymerization induced by chemotactic peptide and concanavalin A in rat neutrophils.

Changes in the state of actin in rat neutrophils were studied after chemotactic peptide and concanavalin A stimulation by using the DNase I inhibition assay. Actin polymerization occurred within seconds after stimulation with F-Met-Leu-Phe and concanavalin A. Pretreatment of cells with cytochalasin D prevented chemotactic peptide-induced actin polymerization. The addition of F-Met-Leu-Phe to lysed cells did not produce any change in actin state. These data offer strong evidence for receptor-induced actin polymerization and support the models implicating actin microfilament formation as a crucial event in cell activation. The observations on platelets, lymphocytes, neutrophils, and islets of Langerhans from different species suggest that actin polymerization might be a universal intracellular event accompanying cell surface receptor perturbation in eukaryotic cells.

Actins↗